1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to a method for detecting or screening for mutations or heterozygosity involving as little as one base change or a single base addition to, or deletion from, the wild-type DNA sequence, as well as methods for removing mismatch-containing DNA from batches of amplified DNA.
2. Description of the Background Art
Progress in human molecular and medical genetics depends on the efficient and accurate detection of mutations and sequence polymorphisms, the vast majority of which results from single base substitutions and small additions or deletions. Assays capable of detecting the presence of a particular mutation or mutant nucleic acid sequence in a sample are therefore of substantial importance in the prediction and diagnosis of disease, forensic medicine, epidemiology and public health. Such assays can be used, for example, to detect the presence of a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a genetic disease. The ability to detect a mutation has taken on increasing importance in early detection of cancer or discovery of susceptibility to cancer with the discovery that discrete mutations in cellular oncogenes can result in activation of that oncogene leading to the transformation of that cell into a cancer cell (Nishimura, S. et al., Biochem. J. 243:313-327 (1987); Bos, J. L., Cancer Res. 49:4682-4689 (1989)).
The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity as well as complexity and cost. Hence, it would be highly desirable to develop more sensitive as well as simple and relatively inexpensive assays for detection of alterations in DNA.
Nucleic acid detection assays can be based on any of a number of characteristics of a nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents such as enzymes (Kourilsky et al., U.S. Pat. No. 4,581,333), radioisotopes (Falkow et al., U.S. Pat. No. 4,358,535; Berninger, U.S. Pat. No. 4,446,237), fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EP 119448), and the like.
Most methods devised to attempt to detect genetic alterations consisting of one or a few bases involve hybridization between a standard nucleic acid (DNA or RNA) and a test DNA such that the mutation is revealed as a mispaired or unpaired base in a heteroduplex molecule. Detection of these mispaired or unpaired bases has been accomplished by a variety of methods. Mismatches have been detected by means of enzymes (RNaseA, MutY) which cut one or both strands of the duplex at the site of a mismatch (Myers, R. M. et al., Cold Spring Harbor Symp. Quant. Biol. 51:275-284 (1986); Gibbs, R. et al., Science 236:303-305 (1987); Lu, A. S. et al., 1992, Genomics 14:249-255 (1992)). Duplexes without mismatches are not cut. By using radioactively labeled nucleic acid fragments to anneal to a test DNA, it is possible to use these enzymes to generate specific size fragments when a mutation is present in the test DNA. The fragments are distinguished from uncut fragments by means of polyacrylamide gel electrophoresis (PAGE). The major problems with these methods are that they require the use of RNA (RNase method) or have the ability to detect only a limited number of mismatches (MutY method).
Mismatch-containing DNA duplexes have also been distinguished from perfectly matched duplexes by means of denaturing gel electrophoresis. In this system, duplexes are run in PAGE in a denaturing gradient under conditions where mismatch-containing DNA denatures more readily than the identical duplex lacking a mismatch, such that the two kinds of duplexes migrate differently. This method, while sensitive and accurate, is extremely laborious and requires a high level of technical sophistication.
Two other methods of mutation detection depend on the failure to extend or join fragments of DNA when mismatches are present. Both require the use of standard DNA oligonucleotides that end precisely at the site of the mutation in question such that, when annealed to test DNA, it is the last base of the oligonucleotide which is mismatched. Mismatch detection depends either on (a) the inability of DNA polymerase to extend an oligonucleotide with a mismatched terminal base or (b) the inability of DNA ligase to join two oligonucleotides when there is a mismatch at the joint between them. Fragment length is determined by gel electrophoresis. Presence of longer fragments than the input oligonucleotides indicates that a mismatch, i.e., mutation, was not present in the test DNA. These methods are also somewhat laborious, require that the exact location of the mutation be known and are difficult to interpret when the sample DNA is heterozygous for the mutation in question. Therefore, they are not practical for use in screening for polymorphisms.
A chemical method for cleavage of mismatched DNA (Cotton, R. G. et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1988); Cotton, R. G., Nuc. Acids Res 17:4223-4233 (1989)) is based on chemical cleavage at a mismatch site in a DNA--DNA heteroduplex, using a number of agents, in particular osmium tetroxide and hydroxylamine. DNA probes are prepared by restriction enzyme cleavage of DNA of interest. Plasmid DNA containing the sequence of interest is hybridized to labeled probe DNA (either end-labeled or internally labeled with .sup.32 P). Hydroxylamine chemically modifies mismatched cytosines; osmium tetroxide modifies mismatched thymines. Piperidine is then used to cleave the DNA at the modified sites. PAGE and autoradiography are then used to identify the cleavage products. This method is said to have the advantage of detecting all possible single base pair mismatches because it results in cleavage at a matched base pair in the vicinity of a mismatch.
Publications from Caskey's laboratory (Caskey, C. T. et al., European Patent Publication 333,465 (Sep. 20, 1989); Grompe, M. et al., Proc. Natl. Acad. Sci. USA 86:5888-5892 (1989)) disclose a method for localizing a mutation which utilizes PCR-amplified cDNA as a source of template for the mismatch cleavage reaction. This technique was successfully applied in studying ornithine transcarbamoylase deficiency patients to map mutations.
Kung et al., U.S. Pat. No. 4,963,658, discloses detection of single stranded DNA (ssDNA) by binding with a high-affinity ssDNA-binding protein, such as a topoisomerase or a DNA unwinding protein which itself can be bound to a label such as .beta.-D-galactosidase.
Wagner et al. PCT publication WO93/02216 shows that E. coli MutS, in solution, detects G//T, G//G and A//C mismatches as well as a frameshift mutation of +1 base. There is no disclosure of using immobilized MutS in this method.
(The symbol "//" is used hereinafter with nucleotide base designations to indicate mispaired bases. Properly paired bases are designated with a ":" for example G:C.)
Lishanskaya et al. (Human Genet. 51, (4 Suppl):A385, abstract #1517 (1992)) is an abstract disclosing the detection of heterozygotes for the amplified CFTR (cystic fibrosis) gene. The method was not shown to be useful for detecting single base mismatches, or mispairings of 1, 2 or 4 bases. Rather, the reference disclosed detection of a mutation causing a three base pair deletion using a gel mobility band-shift assay to detect MutS-heteroduplex complexes but did not suggests immobilizing a MBP.
Lishanski, A. et al., Proc. Natl. Acad. Sci. USA 91:2674-2678 (1994 March) reveals detection of frame shift mutations and A//C, G//T, G//A and T//C mismatches (most convincingly G//T) using MutS. The method worked poorly with duplexes containing single base pair mismatches. At the time that this paper was published, after the filing date of the priority application of this application, the authors were still trying to develop improved detection methods which utilized the methods that the present inventor had already invented and disclosed.
A number of publications describe methods for studying certain DNA binding proteins, in particular sequence-specific transcription factors, by electrophoretic separation (generally under denaturing conditions) of proteins, blotting ("Southwestern blots") or spotting of the separated proteins onto nitrocellulose filters, and probing of the filters with labeled DNA or oligonucleotide preparations. See, for example, Bowen, B. et al., Nucl. Acids Res. 8:1-20 (1979); Miskimins, W. K. et al., Proc. Natl. Acad. Sci. USA 82:6741-6744 (1985); Keller, A. D. et al., Nucl. Acids Res. 19:4675-80 (1991); Norby, P. L. et al., Nucl. Acids Res. 20:6317-6321 (1992)). Such initially denatured proteins must be renatured to perform their DNA binding functions; many proteins cannot be successfully renatured.
Similar approaches have been used to screen expression libraries by preparing protein replica filters and probing them with labeled DNA or oligonucleotides (Singh et al., Cell 52:415-423 (1988); Singh et al., BioTechniques 7:253-261 (1989); Vinson et al., Genes & Devel. 2:801-806 (1988)). Oliphant, A. R. et al. (Molec. Cell. Biol. 9:2944-2949 (1989)) reported use of a sequence-specific DNA binding protein (yeast GCN4 transcriptional activator) coupled to Sepharose to select DNA molecules containing binding sites for this protein from random sequence oligonucleotides.
Interactions of DNA with immobilized E. coli single stranded DNA-binding protein (SSB) has been reported (Perrino, F. W. et al., J. Biol. Chem. 263:11833-11839 (1988)) and is reviewed in Meyer, R. R. et al. (Microbiol. Rev. 54:342-380 (1990)).
3. Mismatch Repair Systems and Mismatch Binding Proteins
DNA mismatch repair systems employ a family of proteins including proteins which recognize and bind to mismatch-containing DNA, which are designated mismatch binding proteins (MBPs). For reviews, see Radman, M. et al., Annu. Rev. Genet. 20:523-538 (1986); Radman, M. et al., Sci. Amer., August 1988, pp. 40-46; Modrich, P., J. Biol. Chem. 264:6597-6600 (1989)). The MutS protein was identified as such a component of the E. coli mismatch repair system. See, for example, Lahue, R. S. et al., Science 245:160-164 (1989); Jiricny, J. et al., Nucl. Acids Res. 16:7843-7853 (1988); Su, S. S. et al., J. Biol. Chem. 263:6829-6835 (1988); Lahue, R. S. et al., Mutat. Res. 198:37-43 (1988); Dohet, C. et al., Mol. Gen. Genet. 206:181-184 (1987); and Jones, M. et al., Genetics 115:605-610 (1987). Analogous proteins are known in other bacterial species including MutS in Salmonella typhimurium (Lu, A. L. et al., Genetics 118:593-600 (1988); Haber L. T. et al., J. Bacteriol. 170:197-202 (1988); Pang, P. P. et al., J. Bacteriol. 163:1007-1015 (1985)) and the hexA protein of Streptococcus pneumoniae (Priebe S. D. et al., J. Bacteriol. 170:190-196 (1988); Haber et al., supra).
Using a gel-shift assay (as well as a filter-binding assay), Jiricny et al. (supra) reported detection of G//T and G//A mismatches, whereas binding to an A//C mismatch was weak and C//C was undetectable.
Su et al. (supra) used a nuclease protection assay to detect binding of MutS to DNA. This reference showed that all the examined mismatches were bound, with binding to A//C, G//A, T//C and G//T mismatches being stronger than binding to A//G, C//T, and C//C mismatches.
Purified MutS protein binds DNA containing mispaired bases, but does not bind DNA without mismatches or single-stranded DNA. The MutS-DNA interaction does not result in any degradation or modification of the DNA. None of the above references disclose the possibility of using a MBP or immobilized MBP as part of a mutation detection assay or for purposes of removing mismatched DNA from amplified DNA samples.
The literature concerning DNA repair suggests that all mismatches are not repaired with equal efficiency in vivo (Dohet, C. et al., Proc. Natl. Acad. Sci. USA 82:503-505 (1985). If poorly-repaired mismatches were created as frequently as well-repaired mismatches, one would not observe the 100-1000-fold increase in mutation rate in mutant cells lacking MutS function. Therefore, the poorly-recognized mismatches, in particular C//C (Dohet et al., supra, Jiricny et al., supra), must not be created as frequently as other well-recognized mismatches, since 99.9% of created mutations are repaired. Hence, MutS must bind to mismatched DNA (and induce the repair process) for virtually any error made by a polymerase enzyme.
With the recent discoveries that mutations in genes coding for MBPs can lead to predisposition to may cancers, particularly colon cancers, there is an increasing interest in determining whether such proteins are present in tumor cells. These MBPs, generally homologs of the bacterial protein MutS, are referred to as MutS homologs (MSH) and are key elements of the mismatch repair system. Individuals heterozygous for a mutation in the human MutS homolog, hMSH2, are predisposed to a form of colon cancer (hereditary non-polyposis colon cancer or HNPCC) (Leach, S. S. et al., Cell 75:1212-1225 (1993); Fishel, R. et al., Cell 75:1027-1029 (1993)). Although the somatic cells of these individuals have normal levels of mismatch repair, HNPCC tumors have been found to be deficient in mismatch repair and have lost function of the homologous copy of the hMSH2 gene, generally by a second mutation Parsons, R. et al., Cell 75:1227-1236 (1993)). HNPCC tumors were originally characterized by the presence of microsatellite instability, also known as the replication error or "RER" phenotype. However, all RER tumors are not necessarily hMSH2 deficient. Mutations in other mismatch repair genes, in particular homologs of the bacterial mutL gene (mlh and pms), can also produce an RER phenotype (Papadopoulos, N. et al., Science 263:1625-1629 (1994); Bronner, E. C. et al., Nature 368:258-261 (1994)). Defects in other cellular functions may also give an RER phenotype (Bhattacharya, N. P. et al., Proc. Natl. Acad. Sci. USA 91:6319-6323 (1994)).
Current methods for determining if a tumor cell line is deficient in mismatch binding activity include in vitro mismatch repair assays and sequencing. Mismatch repair assays are somewhat laborious and cannot distinguish MSH deficiencies from MLH deficiencies unless complementation assays are performed. Sequencing is extremely laborious because the homologous chromosome sequences must be separately cloned and sequenced to distinguish heterozygotes from homozygotes. In addition, neutral polymorphisms, i.e., sequence differences between the homologs which do not affect protein function, can make interpretation of sequencing results difficult.
In response to these deficiencies in the art, the present inventor provides a simple, rapid and inexpensive assay for activity of a MBP in a test sample which does not require the use of gels, sequencing or radioactivity. The assay is based upon the immobilized mismatch binding protein mutation detection assay.
4. Triplet Repeats
A growing number of diseases is known to be associated with the expansion of "triplet" or trinucleotide repeat sequences (Trottier, Y. et al., Current Biology 3:783-786 (1993); Bates, G. et al., Bioessays 16:277-284 (1994); Kawaguchi, Y. et al., Nature Genetics 8:221-227 (1994)). In each of these diseases, the size of the repeat block directly correlates with, and thereby "anticipates" the severity and age of the onset of the disease. Some diseases are correlated with small increases in the size of the repeat block, for example, Huntington's disease. spino-cerebellar ataxia type I, spinal and bulbar muscular atrophy, Machado-Joseph disease and dentatorubralpallidoluysian atrophy. Other diseases involve up to 100-fold expansions of the normal block, such as fragile X type A, fragile X type E and myotonic dystrophy. In general, it appears that, if the repeated motif is short, for example, &lt;10 nucleotides, and the repeat block is shorter than about 0.3 kb, replication slippage is the principal mechanism of instability (Kunkel, T. A., Nature 365:207-208 (1994)). If the repeat block is greater than 0.3 kb (the minimum size for efficient homologous DNA interactions), recombination and/or DNA methylation can affect the size and the sequence of such repeats.
"Satellite DNA" is a term used to describe highly repetitive sequences that are clustered together, and may consist of tandem repeats of a simple sequence. These range in size, with shorter sequences being termed "minisatellite" and yet shorter sequences described as "microsatellite." The most prevalent form of human microsatellite DNA comprises small expansions and contractions of CA.sub.n repeat blocks. These occur very frequently as a result of replication slippage in mismatch repair-deficient HNPCC tumor cells (Bronner, E. C. et al., Nature 368, 258-261 (1994)).
DNA methylation is found in the CGG repeats of fragile X syndrome (Trottier, Y. et al., Current Biology 3:783-786 (1993); Bates, G. et al., Bioessays 16:277-284 (1994); Radman, M. et al. In: GENOME ANALYSIS, vol 7 (Genome Rearrangement and Stability) eds. K. E. Davies et al., pp. 139-152 (1993)). Changes in "minisatellite" size (polymorphic repeat blocks several kb in length) and in DNA sequence occur by gene conversion (Jefferys, A. J. et al., Nature Genetics 6:136-145 (1994)). However, the mechanism of the rapid and large expansions of CGG/CCG and CAG/CTG repeats is unknown.
The present invention is designed in part to detect the presence of such expanded triplet repeat blocks for diagnosing diseases associated with such repeats.